Immunoassays are either currently in use, or planned for use, in detecting infectious disease such as Streptococcus, sexually transmitted diseases such as herpes viruses or chlamydia, and/or pregnancy via the detection of hCG (human chorionic gonadotropin). Drugs are also detectable. Appropriate devices have been developed to run such assays either at a doctor's office or the patient's home, using samples taken from the patient, such as urine. For example, useful devices are described in commonly owned U.S. Ser. No. 98,248 filed by Charles C. Hinckley et al on Sept. 18, 1987, entitled "Sliding Valve For Vent of Liquid Collecting Compartment." The details of that application are expressly incorporated herein by reference.
As described in the aforesaid application, such devices can include one or more chambers containing some of the reagents, each chamber having a sloping side wall that forms a well. At the bottom of each well is a membrane that is porous enough to pass liquid through, but constructed to retain an immunoproduct produced when the analyte in question is present in the patient's sample. The immunoreagents are described in that application as being coated onto the chamber side wall or added by the user. No details are given as to how a coating is to be applied, nor is it stated therein whether the coating is to be a uniform coating or in discrete locations only.
In some assays, such as the pregnancy assay for hCG, the reagents that are pre-incorporated in the chamber include biotinylated antibody for the antigen that is the analyte, and a buffer. The buffer is needed because patient's sample, for example, urine, comes in such a wide range of pH, and the assay works optimally at a pH about 6 to 8. For ease in manufacturing, such buffer is preferably deposited as an essentially fully saturated solution at about 50 wt % concentration, in about 8 .mu.l, to permit the use of small sized drops.
In the hCG assay, at least some of the biotinylated antibody and the buffer need to be deposited on the side wall of the chamber well, rather than on the membrane, because the reaction between biotin and avidin should not take place before patient's sample is added, and certainly not when the device is manufactured. Otherwise the biotinylated antibody is not free to migrate through a solution standing above the membrane to complex with available antigen.
Therefore, at least some of the biotinylated antibody must be deposited and kept on the sloping side wall, until dried.
In addition, some of the buffer is not deposited onto the membrane because (1) the pores of the membrane are too few to retain all the buffer, and (2) even if it could be so retained during manufacturing, much of the buffer so located will wash through too early in the incubation stage during use, if it is initially located in the membrane. That is, a patient sample is designed to "stand" above the membrane for incubation. However, the initial wetting of the membrane by the patient sample causes immediate inadvertent wash through of any reagent in, but not bound to, the membrane. In that case, the buffer would not be retained in the well above the membrane where it is needed to maintain the pH of the patient sample for the antibody complexing reaction.
Nevertheless, prior to this invention it has not been readily apparent how to merely deposit or place liquid containing reagents, onto the sloping side wall without that liquid pouring down onto the membrane instead of drying in place. Cementation of liquid drops to the side wall, followed by drying, is of course inappropriate, in that the reagent must be capable of re-dissolving when patient liquid is added. The problem is particularly aggrevated by the fact that the preferred material for the wall construction--a dyed polystyrene--has a lower tolerance for drop adherence than other wall materials, for example polystyrene not containing a dye preincorporated into it.
It would be conceivable to construct the well with a horizontal shelf in the wall that provides a level platform for location of the drops. One difficulty with such an arrangement is that it becomes very difficult to force all the liquid off such a shelf, and down onto the membrane where separation of bound and free labeled antibody occurs.
Alternatively, the slope of the wall could be rendered more gradual, to the point at which almost any volume drop will stick to any surface. This is not viable either, as a slight decrease in the wall slope drastically increases the amount of sample volume that has to be used for a given exposed area of membrane. In addition, the area of the membrane can not be reduced much, since it then becomes difficult to detect a color change on the membrane.
Thus, for the reasons given above, the sloping wall is generally restricted to an angle of at least 60.degree., for example about 65.degree., and the exposed membrane surface area is about 0.2 cm.sup.2.